The establishment and maintenance of cellular asymmetry is a means of generating a differential protein composition within a continuous cytoplasm. Localization of mRNA is a means of achieving this asymmetry that is widely used across organisms. In the budding yeast Saccharomyces cerevisiae, mating type switching is tightly controlled by the asymmetric accumulation of the ASH1 gene product. Events that occur at every stage of ASH1 production from its transcription, packaging and transport to the bud tip to its degradation are imperative for efficacy of mating type switching. Our previous work has led to the identification of factors such as PufGp that exert translational control over ASH1 mRNA during transport to the bud tip, as well as solving the crystal structure of She2p, that revealed a novel RNA-binding motif. This grant will expand on the strong foundation that has been established in our lab for the study of RNA localization, allowing us to further isolate and characterize factors that affect ASH1 localization at every stage of its life cycle. At the same time we will continue projects that broaden the scope of our understanding of gene expression to include global studies that define and characterize the variation inherent in transcriptional activation as well as the detailed analysis of a regulatory loop that spans the entire cell cycle. The nature of our analysis, which includes the refinement and development of microscopy and computer analysis tools allows for a population analysis with a single-cell resolution that offers a novel perspective on gene expression. Our capability to innovate and integrate technology with fundamental laboratory techniques will allow us ultimately to follow a single mRNA from transcription, to nuclear export, to translation and its subsequent degradation, all in real time, in living cells.